PROJECT SUMMARY We will develop a new approach for comprehensive antibody repertoire analysis that enables rapid functional identification of broadly neutralizing antibodies (bNAbs) from HIV-infected patients and vaccine recipients. Available technologies for anti-HIV-1 antibody discovery rely on laborious single B cell cloning and antibody expression, which provide limited antibody functional information are unable to characterize more than a few hundred antibodies in a single sample. In this project, we will utilize a recently invented antibody yeast display platform to immortalize natively paired heavy+light antibodies repertoires en masse and screen them for potent antibodies targeting HIV-1 antigens. We will validate our techniques for screening immune repertoires by discovering HIV bNAbs from clinical samples derived from the Center for AIDS Programme of Research in South Africa (CAPRISA) longitudinal cohort. First, we will identify epitope-specific antibodies that target known HIV-1 vulnerable epitopes for rapid HIV-1 bNAb discovery. We will apply native antibody screening in yeast display to annotate CAPRISA patient immune repertoires and identify the entire scope of antibodies targeting known bNAb epitopes. Next, we will screen CAPRISA immune repertoires to determine the affinity of anti-HIV-1 antibodies in the repertoire and to identify high-affinity broad antibodies as bNAb lead candidates. Finally, we will establish a system that enables rapid antibody secretion and HIV-1 neutralization assays to accelerate the timeline for molecular validation of bNAb lead candidates. Successful completion of this project will deliver a comprehensive overview of anti-HIV-1 B cell responses in CAPRISA patient samples and discover new HIV bNAbs for follow-up analysis. It is expected that future studies will leverage these technologies to advance vaccine and therapeutic discovery against other infectious diseases. Our long-term objective is to apply advanced antibody screening technologies to enhance understanding of anti-HIV B cell function and to accelerate vaccine design against HIV-1 and other pathogens of major public health importance.